Myeloid-derived suppressor cells and tolerogenic dendritic cells are distinctively induced by PI3K and Wnt signaling pathways.

Imbalanced immune responses are a prominent hallmark of cancer and autoimmunity. Myeloid cells can be overly suppressive, inhibiting protective immune responses or inactive not controlling autoreactive immune cells. Understanding the mechanisms that induce suppressive myeloid cells, such as myeloid-derived suppressor cells (MDSCs) and tolerogenic dendritic cells (TolDCs), can facilitate the development of immune-restoring therapeutic approaches. MDSCs are a major barrier for effective cancer immunotherapy by suppressing antitumor immune responses in cancer patients. TolDCs are administered to patients to promote immune tolerance with the intent to control autoimmune disease. Here, we investigated the development and suppressive/tolerogenic activity of human MDSCs and TolDCs to gain insight into signaling pathways that drive immunosuppression in these different myeloid subsets. Moreover, monocyte-derived MDSCs (M-MDSCs) generated in vitro were compared to M-MDSCs isolated from head-and-neck squamous cell carcinoma patients. PI3K-AKT signaling was identified as being crucial for the induction of human M-MDSCs. PI3K inhibition prevented the downregulation of HLA-DR and the upregulation of reactive oxygen species and MerTK. In addition, we show that the suppressive activity of dexamethasone-induced TolDCs is induced by ß-catenin-dependent Wnt signaling. The identification of PI3K-AKT and Wnt signal transduction pathways as respective inducers of the immunomodulatory capacity of M-MDSCs and TolDCs provides opportunities to overcome suppressive myeloid cells in cancer patients and optimize therapeutic application of TolDCs. Lastly, the observed similarities between generated- and patient-derived M-MDSCs support the use of in vitro-generated M-MDSCs as powerful model to investigate the functionality of human MDSCs.

pH and ROS Responsiveness of Polymersome Nanovaccines for Antigen and Adjuvant Codelivery: An In Vitro and In Vivo Comparison.

The antitumor immunity can be enhanced through the synchronized codelivery of antigens and immunostimulatory adjuvants to antigen-presenting cells, particularly dendritic cells (DCs), using nanovaccines (NVs). To study the influence of intracellular vaccine cargo release kinetics on the T cell activating capacities of DCs, we compared stimuli-responsive to nonresponsive polymersome NVs. To do so, we employed "AND gate" multiresponsive (MR) amphiphilic block copolymers that decompose only in response to the combination of chemical cues present in the environment of the intracellular compartments in antigen cross-presenting DCs: low pH and high reactive oxygen species (ROS) levels. After being unmasked by ROS, pH-responsive side chains are exposed and can undergo a charge shift within a relevant pH window of the intracellular compartments in antigen cross-presenting DCs. NVs containing the model antigen Ovalbumin (OVA) and the iNKT cell activating adjuvant α-Galactosylceramide (α-Galcer) were fabricated using microfluidics self-assembly. The MR NVs outperformed the nonresponsive NV in vitro, inducing enhanced classical- and cross-presentation of the OVA by DCs, effectively activating CD8+, CD4+ T cells, and iNKT cells. Interestingly, in vivo, the nonresponsive NVs outperformed the responsive vaccines. These differences in polymersome vaccine performance are likely linked to the kinetics of cargo release, highlighting the crucial chemical requirements for successful cancer nanovaccines.

Insertion of atypical glycans into the tumor antigen-binding site identifies DLBCLs with distinct origin and behavior.

Glycosylation of the surface immunoglobulin (Ig) variable region is a remarkable follicular lymphoma-associated feature rarely seen in normal B cells. Here, we define a subset of diffuse large B-cell lymphomas (DLBCLs) that acquire N-glycosylation sites selectively in the Ig complementarity-determining regions (CDRs) of the antigen-binding sites. Mass spectrometry and X-ray crystallography demonstrate how the inserted glycans are stalled at oligomannose-type structures because they are buried in the CDR loops. Acquisition of sites occurs in ∼50% of germinal-center B-cell-like DLBCL (GCB-DLBCL), mainly of the genetic EZB subtype, irrespective of IGHV-D-J use. This markedly contrasts with the activated B-cell-like DLBCL Ig, which rarely has sites in the CDR and does not seem to acquire oligomannose-type structures. Acquisition of CDR-located acceptor sites associates with mutations of epigenetic regulators and BCL2 translocations, indicating an origin shared with follicular lymphoma. Within the EZB subtype, these sites are associated with more rapid disease progression and with significant gene set enrichment of the B-cell receptor, PI3K/AKT/MTORC1 pathway, glucose metabolism, and MYC signaling pathways, particularly in the fraction devoid of MYC translocations. The oligomannose-type glycans on the lymphoma cells interact with the candidate lectin dendritic cell-specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), mediating low-level signals, and lectin-expressing cells form clusters with lymphoma cells. Both clustering and signaling are inhibited by antibodies specifically targeting the DC-SIGN carbohydrate recognition domain. Oligomannosylation of the tumor Ig is a posttranslational modification that readily identifies a distinct GCB-DLBCL category with more aggressive clinical behavior, and it could be a potential precise therapeutic target via antibody-mediated inhibition of the tumor Ig interaction with DC-SIGN-expressing M2-polarized macrophages.

Human type 1 and type 2 conventional dendritic cells express indoleamine 2,3-dioxygenase 1 with functional effects on T cell priming.

Dendritic cells (DCs) are key regulators of the immune system that shape T cell responses. Regulation of T cell induction by DCs may occur via the intracellular enzyme indoleamine 2,3-dioxygenase 1 (IDO), which catalyzes conversion of the essential amino acid tryptophan into kynurenine. Here, we examined the role of IDO in human peripheral blood plasmacytoid DCs (pDCs), and type 1 and type 2 conventional DCs (cDC1s and cDC2s). Our data demonstrate that under homeostatic conditions, IDO is selectively expressed by cDC1s. IFN-γ or TLR ligation further increases IDO expression in cDC1s and induces modest expression of the enzyme in cDC2s, but not pDCs. IDO expressed by conventional DCs is functionally active as measured by kynurenine production. Furthermore, IDO activity in TLR-stimulated cDC1s and cDC2s inhibits T cell proliferation in settings were DC-T cell cell-cell contact does not play a role. Selective inhibition of IDO1 with epacadostat, an inhibitor currently tested in clinical trials, rescued T cell proliferation without affecting DC maturation status or their ability to cross-present soluble antigen. Our findings provide new insights into the functional specialization of human blood DC subsets and suggest a possible synergistic enhancement of therapeutic efficacy by combining DC-based cancer vaccines with IDO inhibition.

Multiscale imaging of therapeutic anti-PD-L1 antibody localization using molecularly defined imaging agents.

BACKGROUND: While immune checkpoint inhibitors such as anti-PD-L1 antibodies have revolutionized cancer treatment, only subgroups of patients show durable responses. Insight in the relation between clinical response, PD-L1 expression and intratumoral localization of PD-L1 therapeutics could improve patient stratification. Therefore, we present the modular synthesis of multimodal antibody-based imaging tools for multiscale imaging of PD-L1 to study intratumoral distribution of PD-L1 therapeutics. RESULTS: To introduce imaging modalities, a peptide containing a near-infrared dye (sulfo-Cy5), a chelator (DTPA), an azide, and a sortase-recognition motif was synthesized. This peptide and a non-fluorescent intermediate were used for site-specific functionalization of c-terminally sortaggable mouse IgG1 (mIgG1) and Fab anti-PD-L1. To increase the half-life of the Fab fragment, a 20 kDa PEG chain was attached via strain-promoted azide-alkyne cycloaddition (SPAAC). Biodistribution and imaging studies were performed with 111In-labeled constructs in 4T1 tumor-bearing mice. Comparing our site-specific antibody-conjugates with randomly conjugated antibodies, we found that antibody clone, isotype and method of DTPA conjugation did not change tumor uptake. Furthermore, addition of sulfo-Cy5 did not affect the biodistribution. PEGylated Fab fragment displayed a significantly longer half-life compared to unPEGylated Fab and demonstrated the highest overall tumor uptake of all constructs. PD-L1 in tumors was clearly visualized by SPECT/CT, as well as whole body fluorescence imaging. Immunohistochemistry staining of tumor sections demonstrated that PD-L1 co-localized with the fluorescent and autoradiographic signal. Intratumoral localization of the imaging agent could be determined with cellular resolution using fluorescent microscopy. CONCLUSIONS: A set of molecularly defined multimodal antibody-based PD-L1 imaging agents were synthesized and validated for multiscale monitoring of PD-L1 expression and localization. Our modular approach for site-specific functionalization could easily be adapted to other targets.

Regulatory T Cell Depletion Using a CRISPR Fc-Optimized CD25 Antibody.

Regulatory T cells (Tregs) are major drivers behind immunosuppressive mechanisms and present a major hurdle for cancer therapy. Tregs are characterized by a high expression of CD25, which is a potentially valuable target for Treg depletion to alleviate immune suppression. The preclinical anti-CD25 (αCD25) antibody, clone PC-61, has met with modest anti-tumor activity due to its capacity to clear Tregs from the circulation and lymph nodes, but not those that reside in the tumor. The optimization of the Fc domain of this antibody clone has been shown to enhance the intratumoral Treg depletion capacity. Here, we generated a stable cell line that produced optimized recombinant Treg-depleting antibodies. A genome engineering strategy in which CRISPR-Cas9 was combined with homology-directed repair (CRISPR-HDR) was utilized to optimize the Fc domain of the hybridoma PC-61 for effector functions by switching it from its original rat IgG1 to a mouse IgG2a isotype. In a syngeneic tumor mouse model, the resulting αCD25-m2a (mouse IgG2a isotype) antibody mediated the effective depletion of tumor-resident Tregs, leading to a high effector T cell (Teff) to Treg ratio. Moreover, a combination of αCD25-m2a and an αPD-L1 treatment augmented tumor eradication in mice, demonstrating the potential for αCD25 as a cancer immunotherapy.

Three distinct tolerogenic CD14+ myeloid cell types to actively manage autoimmune disease: Opportunities and challenges.

Current treatment for patients with autoimmune disorders including rheumatoid arthritis, multiple sclerosis and type 1 diabetes, often consists of long-term drug regimens that broadly dampen immune responses. These non-specific treatments are frequently associated with severe side effects creating an urgent need for safer and more effective therapy to promote peripheral tolerance in autoimmune diseases. Cell-based immunotherapy may offer an encouraging alternative, where tolerogenic CD14+ myeloid cells are infused to inhibit autoreactive effector cells. In this review, we compared in depth three promising tolerogenic CD14+ candidates for the treatment of autoimmune disease: 1) tolerogenic dendritic cells, 2) monocytic myeloid-derived suppressor cells and 3) CD14+ type 2 conventional dendritic cells. TolDC-based therapy has entered clinical testing whereas evidence from the latter two cell types m-MDSCs and CD14+ cDC2s is predominantly coming from cancer immunology research. These three cell types have distinct cellular properties and immunosuppressive mechanisms offering unique opportunities to be explored. However, these cells differ in stage of development towards immunotherapy each facing additional hurdles. Therefore, we speculate on the potential benefits and risks of these cell types as novel cell-based immunotherapies to control autoimmune disease in patients.

Characterization of Intrinsically Radiolabeled Poly(lactic-co-glycolic acid) Nanoparticles for ex Vivo Autologous Cell Labeling and in Vivo Tracking.

With the advent of novel immunotherapies, interest in ex vivo autologous cell labeling for in vivo cell tracking has revived. However, current clinically available labeling strategies have several drawbacks, such as release of radiolabel over time and cytotoxicity. Poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) are clinically used biodegradable carriers of contrast agents, with high loading capacity for multimodal imaging agents. Here we show the development of PLGA-based NPs for ex vivo cell labeling and in vivo cell tracking with SPECT. We used primary amine-modified PLGA polymers (PLGA-NH2) to construct NPs similar to unmodified PLGA NPs. PLGA-NH2 NPs were efficiently radiolabeled without chelator and retained the radionuclide for 2 weeks. Monocyte-derived dendritic cells labeled with [111In]In-PLGA-NH2 showed higher specific activity than those labeled with [111In]In-oxine, with no negative effect on cell viability. SPECT/CT imaging showed that radiolabeled THP-1 cells accumulated at the Staphylococcus aureus infection site in mice. In conclusion, PLGA-NH2 NPs are able to retain 111In, independent of chelator presence. Furthermore, [111In]In-PLGA-NH2 allows cell labeling with high specific activity and no loss of activity over prolonged time intervals. Finally, in vivo tracking of ex vivo labeled THP-1 cells was demonstrated in an infection model using SPECT/CT imaging.

Dual Site-Specific Chemoenzymatic Antibody Fragment Conjugation Using CRISPR-Based Hybridoma Engineering.

Functionalized antibodies and antibody fragments have found applications in the fields of biomedical imaging, theranostics, and antibody-drug conjugates (ADC). In addition, therapeutic and theranostic approaches benefit from the possibility to deliver more than one type of cargo to target cells, further challenging stochastic labeling strategies. Thus, bioconjugation methods to reproducibly obtain defined homogeneous conjugates bearing multiple different cargo molecules, without compromising target affinity, are in demand. Here, we describe a straightforward CRISPR/Cas9-based strategy to rapidly engineer hybridoma cells to secrete Fab' fragments bearing two distinct site-specific labeling motifs, which can be separately modified by two different sortase A mutants. We show that sequential genetic editing of the heavy chain (HC) and light chain (LC) loci enables the generation of a stable cell line that secretes a dual tagged Fab' molecule (DTFab'), which can be easily isolated. To demonstrate feasibility, we functionalized the DTFab' with two distinct cargos in a site-specific manner. This technology platform will be valuable in the development of multimodal imaging agents, theranostics, and next-generation ADCs.

C-type lectin-like receptor 2 (CLEC-2)-dependent dendritic cell migration is controlled by tetraspanin CD37.

Cell migration is central to evoking a potent immune response. Dendritic cell (DC) migration to lymph nodes is dependent on the interaction of C-type lectin-like receptor 2 (CLEC-2; encoded by the gene Clec1b), expressed by DCs, with podoplanin, expressed by lymph node stromal cells, although the underlying molecular mechanisms remain elusive. Here, we show that CLEC-2-dependent DC migration is controlled by tetraspanin CD37, a membrane-organizing protein. We identified a specific interaction between CLEC-2 and CD37, and myeloid cells lacking CD37 (Cd37-/-) expressed reduced surface CLEC-2. CLEC-2-expressing Cd37-/- DCs showed impaired adhesion, migration velocity and displacement on lymph node stromal cells. Moreover, Cd37-/- DCs failed to form actin protrusions in a 3D collagen matrix upon podoplanin-induced CLEC-2 stimulation, phenocopying CLEC-2-deficient DCs. Microcontact printing experiments revealed that CD37 is required for CLEC-2 recruitment in the membrane to its ligand podoplanin. Finally, Cd37-/- DCs failed to inhibit actomyosin contractility in lymph node stromal cells, thus phenocopying CLEC-2-deficient DCs. This study demonstrates that tetraspanin CD37 controls CLEC-2 membrane organization and provides new molecular insights into the mechanisms underlying CLEC-2-dependent DC migration.This article has an associated First Person interview with the first author of the paper.

Autologous monocyte-derived DC vaccination combined with cisplatin in stage III and IV melanoma patients: a prospective, randomized phase 2 trial.

BACKGROUND: Autologous dendritic cell (DC) vaccines can induce tumor-specific T cells, but their effect can be counteracted by immunosuppressive mechanisms. Cisplatin has shown immunomodulatory effects in vivo which may enhance efficacy of DC vaccination. METHODS: This is a prospective, randomized, open-label phase 2 study (NCT02285413) including stage III and IV melanoma patients receiving 3 biweekly vaccinations of gp100 and tyrosinase mRNA-loaded monocyte-derived DCs with or without cisplatin. Primary objectives were to study immunogenicity and feasibility, and secondary objectives were to assess toxicity and survival. RESULTS: Twenty-two stage III and 32 stage IV melanoma patients were analyzed. Antigen-specific CD8+ T cells were found in 44% versus 67% and functional T cell responses in 28% versus 19% of skin-test infiltrating lymphocytes in patients receiving DC vaccination with and without cisplatin, respectively. Four patients stopped cisplatin because of toxicity and continued DC monotherapy. No therapy-related grade 3 or 4 adverse events occurred due to DC monotherapy. During combination therapy, one therapy-related grade 3 adverse event, decompensated heart failure due to fluid overload, occurred. The clinical outcome parameters did not clearly suggest significant differences. CONCLUSIONS: Combination of DC vaccination and cisplatin in melanoma patients is feasible and safe, but does not seem to result in more tumor-specific T cell responses or improved clinical outcome, when compared to DC vaccination monotherapy.

Eight-Color Multiplex Immunohistochemistry for Simultaneous Detection of Multiple Immune Checkpoint Molecules within the Tumor Microenvironment.

Therapies targeting immune checkpoint molecules CTLA-4 and PD-1/PD-L1 have advanced the field of cancer immunotherapy. New mAbs targeting different immune checkpoint molecules, such as TIM3, CD27, and OX40, are being developed and tested in clinical trials. To make educated decisions and design new combination treatment strategies, it is vital to learn more about coexpression of both inhibitory and stimulatory immune checkpoints on individual cells within the tumor microenvironment. Recent advances in multiple immunolabeling and multispectral imaging have enabled simultaneous analysis of more than three markers within a single formalin-fixed paraffin-embedded tissue section, with accurate cell discrimination and spatial information. However, multiplex immunohistochemistry with a maximized number of markers presents multiple difficulties. These include the primary Ab concentrations and order within the multiplex panel, which are of major importance for the staining result. In this article, we report on the development, optimization, and application of an eight-color multiplex immunohistochemistry panel, consisting of PD-1, PD-L1, OX40, CD27, TIM3, CD3, a tumor marker, and DAPI. This multiplex panel allows for simultaneous quantification of five different immune checkpoint molecules on individual cells within different tumor types. This analysis revealed major differences in the immune checkpoint expression patterns across tumor types and individual tumor samples. This method could ultimately, by characterizing the tumor microenvironment of patients who have been treated with different immune checkpoint modulators, form the rationale for the design of immune checkpoint-based immunotherapy in the future.

Multicore Liquid Perfluorocarbon-Loaded Multimodal Nanoparticles for Stable Ultrasound and 19F MRI Applied to In Vivo Cell Tracking.

Ultrasound is the most commonly used clinical imaging modality. However, in applications requiring cell-labeling, the large size and short active lifetime of ultrasound contrast agents limit their longitudinal use. Here, 100 nm radius, clinically applicable, polymeric nanoparticles containing a liquid perfluorocarbon, which enhance ultrasound contrast during repeated ultrasound imaging over the course of at least 48 h, are described. The perfluorocarbon enables monitoring the nanoparticles with quantitative 19F magnetic resonance imaging, making these particles effective multimodal imaging agents. Unlike typical core-shell perfluorocarbon-based ultrasound contrast agents, these nanoparticles have an atypical fractal internal structure. The nonvaporizing highly hydrophobic perfluorocarbon forms multiple cores within the polymeric matrix and is, surprisingly, hydrated with water, as determined from small-angle neutron scattering and nuclear magnetic resonance spectroscopy. Finally, the nanoparticles are used to image therapeutic dendritic cells with ultrasound in vivo, as well as with 19F MRI and fluorescence imaging, demonstrating their potential for long-term in vivo multimodal imaging.

Dendritic cell subsets digested: RNA sensing makes the difference!

In this issue of Immunity, Luber et al. (2010) report a comprehensive quantitative proteome of in vivo mouse spleen dendritic cell (DC) subsets: a data set of encyclopedic value already revealing that DC subsets exploit different RNA sensors for virus recognition.

Synthetic Semiflexible and Bioactive Brushes.

Polymer brushes are extensively used for the preparation of bioactive surfaces. They form a platform to attach functional (bio)molecules and control the physicochemical properties of the surface. These brushes are nearly exclusively prepared from flexible polymers, even though much stiffer brushes from semiflexible polymers are frequently found in nature, which exert bioactive functions that are out of reach for flexible brushes. Synthetic semiflexible polymers, however, are very rare. Here, we use polyisocyanopeptides (PICs) to prepare high-density semiflexible brushes on different substrate geometries. For bioconjugation, we developed routes with two orthogonal click reactions, based on the strain-promoted azide-alkyne cycloaddition reaction and the (photoactivated) tetrazole-ene cycloaddition reaction. We found that for high brush densities, multiple bonds between the polymer and the substrate are necessary, which was achieved in a block copolymer strategy. Whether the desired biomolecules are conjugated to the PIC polymer before or after brush formation depends on the dimensions and required densities of the biomolecules and the curvature of the substrate. In either case, we provide mild, aqueous, and highly modular reaction strategies, which make PICs a versatile addition to the toolbox for generating semiflexible bioactive polymer brush surfaces.

ICAM3-Fc Outperforms Receptor-Specific Antibodies Targeted Nanoparticles to Dendritic Cells for Cross-Presentation.

Optimal targeting of nanoparticles (NP) to dendritic cells (DCs) receptors to deliver cancer-specific antigens is key to the efficient induction of anti-tumour immune responses. Poly (lactic-co-glycolic acid) (PLGA) nanoparticles containing tètanus toxoid and gp100 melanoma-associated antigen, toll-like receptor adjuvants were targeted to the DC-SIGN receptor in DCs by specific humanized antibodies or by ICAM3-Fc fusion proteins, which acts as the natural ligand. Despite higher binding and uptake efficacy of anti-DC-SIGN antibody-targeted NP vaccines than ICAM3-Fc ligand, no difference were observed in DC activation markers CD80, CD83, CD86 and CCR7 induced. DCs loaded with NP coated with ICAM3-Fc appeared more potent in activating T cells via cross-presentation than antibody-coated NP vaccines. This fact could be very crucial in the design of new cancer vaccines.

Endolysosomal-Escape Nanovaccines through Adjuvant-Induced Tumor Antigen Assembly for Enhanced Effector CD8+ T Cell Activation.

The activation of tumor-specific effector immune cells is key for successful immunotherapy and vaccination is a powerful strategy to induce such adaptive immune responses. However, the generation of effective anticancer vaccines is challenging. To overcome these challenges, a novel straight-forward strategy of adjuvant-induced tumor antigen assembly to generate nanovaccines with superior antigen/adjuvant loading efficiency is developed. To protect nanovaccines in circulation and to introduce additional functionalities, a biocompatible polyphenol coating is installed. The resulting functionalizable nanovaccines are equipped with a pH (low) insertion peptide (pHLIP) to facilitate endolysosomal escape and to promote cytoplasmic localization, with the aim to enhance cross-presentation of the antigen by dendritic cells to effectively activate CD8+ T cell. The results demonstrate that pHLIP-functionalized model nanovaccine can induce endolysosomal escape and enhance CD8+ T cell activation both in vitro and in vivo. Furthermore, based on the adjuvant-induced antigen assembly, nanovaccines of the clinically relevant tumor-associated antigen NY-ESO-1 are generated and show excellent capacity to elicit NY-ESO-1-specific CD8+ T cell activation, demonstrating a high potential of this functionalizable nanovaccine formulation strategy for clinical applications.

Semaphorin 7A Promotes Chemokine-Driven Dendritic Cell Migration.

Dendritic cell (DC) migration is essential for efficient host defense against pathogens and cancer, as well as for the efficacy of DC-based immunotherapies. However, the molecules that induce the migratory phenotype of DCs are poorly defined. Based on a large-scale proteome analysis of maturing DCs, we identified the GPI-anchored protein semaphorin 7A (Sema7A) as being highly expressed on activated primary myeloid and plasmacytoid DCs in human and mouse. We demonstrate that Sema7A deficiency results in impaired chemokine CCL21-driven DC migration in vivo. Impaired formation of actin-based protrusions, resulting in slower three-dimensional migration, was identified as the mechanism underlying the DC migration defect. Furthermore, we show, by atomic force microscopy, that Sema7A decreases adhesion strength to extracellular matrix while increasing the connectivity of adhesion receptors to the actin cytoskeleton. This study demonstrates that Sema7A controls the assembly of actin-based protrusions that drive DC migration in response to CCL21.

Affinity-Based Purification of Polyisocyanopeptide Bioconjugates.

Water-soluble polyisocyanopeptides (PICs) are a new class of synthetic polymers that mimic natural protein-based filaments. Their unique semiflexible properties combined with a length of several hundred nanometers have recently enabled a number of biomedical applications ranging from tissue engineering to cancer immunotherapy. One crucial step toward the further development of PICs for these applications is the efficient and controlled synthesis and purification of PIC-biomolecule conjugates. Considering the large size of PICs and the biomolecules to be conjugated, conjugation reactions do usually not proceed to completion due to steric effects. As a consequence, purification of the reaction mixture is necessary to separate the obtained bioconjugates from unreacted biomolecules. As a direct result of the semiflexible nature of PICs, standard polymer and protein purification methods based on molecular weight have not been successful. Here, we introduce a new affinity-based purification method utilizing biotin as an affinity tag. PICs decorated with a controlled and tunable density of biotin molecules (biotinPICs) were efficiently bound to and eluted from a monoavidin resin in buffered aqueous solution. Using these biotinPICs, two different protein conjugates were synthesized, one carrying the enzyme alkaline phosphatase (PhoA) and the other T-cell activating anti-CD3 antibodies. The resulting biotinPIC-protein conjugates were successfully obtained in high purity (>90%) and without any loss of protein activity. The high purity greatly simplifies the analysis of biotinPIC bioconjugates, such as the determination of the average number of biomolecules conjugated per biotinPIC chain. Most importantly, it allows for the direct and straightforward application of the obtained bioconjugates in the desired applications. The new method developed may further be adapted for the purification of other advanced bioconjugates that are difficult to obtain in high purity with the available standard methods.

Multispectral imaging for highly accurate analysis of tumour-infiltrating lymphocytes in primary melanoma.

AIMS: The quality and quantity of the infiltration of immune cells into tumour tissues have substantial impacts on patients' clinical outcomes, and are associated with response to immunotherapy. Therefore, the precise analysis of tumour-infiltrating lymphocytes (TILs) is becoming an important additional pathological biomarker. Analysis of TILs is usually performed semiquantitatively by pathologists on haematoxylin and eosin-stained or immunostained tissue sections. However, automated quantification outperforms semiquantitative approaches, and is becoming the standard. Owing to the presence of melanin pigment, this approach is seriously hampered in melanoma, because the spectrum of melanin lies close to that of commonly used immunohistochemical stains. Aim of this study is to overcome the technical issues due to the presence of melanin for an automated and accurate quantification of TILs in melanoma. METHODS AND RESULTS: Here, we successfully applied a novel multispectral imaging (MSI) technique to enumerate T cells in human primary melanomas. This microscopy technique combines imaging with spectroscopy to obtain both quantitative expression data and the tissue distributions of different cellular markers. We demonstrate that MSI allows complete and accurate analysis of TILs, successfully avoiding the blurring of images by melanin pigments, in whole tissue slide primary melanoma lesions, which could otherwise not be accurately detected by conventional digital image methodologies. CONCLUSIONS: Our study highlights the potential of MSI for accurate assessment of immune cell infiltrates, including those in notoriously difficult tissues, such as pigmented melanomas. Quantification of tumour infiltration by different immune cell types is crucial in the search for new biomarkers to predict patient responses to immunotherapies. Our findings show that this innovative microscopy technique is an important extension of the armamentarium of pathologists.